Project Summary/Abstract Sulfated glycosaminoglycan (GAG) carbohydrates represent one of the most structurally diverse groups of biomolecules and a comprehensive understanding of their biological structure-function relationships has yet to be achieved. Unlike other biomolecules such as DNA/RNA and proteins that are synthesized based upon a template, GAG biosynthesis is the result of the cumulative actions of a series of biosynthetic enzymes that produce a dynamic, polydisperse mixture. The composition of this mixture is dependent on factors such as organism age, developmental or disease state, and tissue of origin. Although this diversity presents a daunting analytical challenge, significant progress has been made in the field through attempts to isolate and characterize GAGs ranging from intact polysaccharides to enzymatically prepared oligosaccharides and disaccharides. The most widespread approach is to profile GAG disaccharides through separation (HPLC, UHPLC, HILIC, CE) and detection (UV, fluorescence, mass spectrometry (MS)). Domains can be characterized for structure-function studies by combining these techniques into hyphenated methods (e.g. LC- MS). Even though disaccharide analysis is the most widely utilized method for GAG analysis, it is unable to provide the structural motifs in GAGs that participate in protein binding without complementary enzymology and domain modeling. We propose to develop a high throughput CE-MS/MS platform for GAG structural characterization that will enable widespread engagement from the glycoscience community by incorporating CE for GAG oligosaccharide separation, a novel CE-MS interface technology, and robust tandem mass spectrometry approaches.